of 12
1
Supplementary
Information
Supplementary Text
De novo transcriptome assembly and reconstruction of
metagenome
-
assembled genomes
De novo assembly of the transcripts independently confirmed that the SRB partners of
ANME were inactive
in the presence of AQDS
. The
majority of
the expressed genes in the active
conditions (
Sulfate, AQDS, and AQDS+Sulfate
)
were associated with the
Methanosarcinales
(
Supplementary
Figure
6
A
)
,
the order of which all families of ANME
-
2 belong
[1]
.
Expressed
genes associated with the
Deltaproteobacteria
(now
known
as the
Desulfobacterota
[2]
),
which
harbor
t
he dominant SRB
lineage
SEEP
-
SRB1
in
our sediment
sample,
were
a large portion of
the total transcripts when sulfate was
actively consumed
as the electron acceptor
for AOM
, but
significantly decreased
in the
AQDS or AQDS+Sulfate
conditions
(
Supplementary
Figure
6
A
).
With the caveat that not all of the transcripts could be confidently assigned to order or class
levels, our taxonomic analysis of the transcripts support that ANME and SRB are the most
abundant active members in the seep sediment incubations. A
ssignment of mRNA transcripts to
known
metabolic pathways
revealed
the majority
were
assigned to methane metabolism
, a
pathway that was highly expressed
in all conditions relative to the no electron acceptor control
(
Supplementary
Figure
6
B). Transcripts
a
ssigned to
sulfur metabolism
were
a
bundant
in the
Sulfate condition
, but
significantly lower in AQDS, AQDS+Sulfate and
no electron acceptor
control
(None)
conditions (
Supplementary
Figure
6
B).
We reconstructed metagenome
-
assembled genomes (MAGs) that rep
resent the core
genomes of the dominant ANME (ANME
-
2a and ANME
-
2c) and SRB lineages (SEEP
-
SRB1a
and SEEP
-
SRB1
g
) from the high
-
coverage reads of bulk sediment metagenome of the same
sediment sample used for the metatranscriptomics analysis (Supplementary Te
xt
,
Supplementary
Table
3
). Given the inherent high strain
-
level heterogeneity among co
-
occurring ANME in seep
sediments, the reconstructed MAGs are smaller than the expected genome size of 3
-
4 Mb based
on previous studies
[3
5]
and likely represent the core rather than the variable component of the
genome between strains. The
SEEP
-
SRB1
g
MAG is notably smaller (only
0.6 Mb) and
estimated at only 36% genome completeness, whereas the MAGs of ANME
-
2a, ANME
-
2c and
SEEP
-
SRB1a estimated at ~ 90% genome completeness (Supplementary Table
3
). Together,
these 4 MAGs account for the majority of the transcripts under our experime
ntal conditions, with
the two ANMEs accounting for 62% and the two SRBs accounting for 16% of the non
-
rRNA
transcripts on average recovered in the Sulfate
condition
(Supplementary Figure
7
). Previous
transcriptomic studies of AOM consortia also reported ANME accounting for a greater
proportion of the expressed genes than SRB
[4]
, which is in contrast to the presumed anabolic
activity of ANME and SRB measured from nanoSIMS
-
based single cell
15
N
-
ammon
ium
assimilation data (Figure
4
). Compared to the
no electron acceptor control
condition, both
ANME
-
2a and ANME
-
2c showed an upregulation in transcripts with the addition of either
sulfate or AQDS as the electron acceptor, whereas
SEEP
-
SRB1a and
SEEP
-
SRB1
g
sho
wed
transcriptional upregulation in the Sulfate condition only (Figure
5
).
2
Supplementary Tables and Figures
Supplementary Figure 1. Sediment microcosms incubated with N
2
/CO
2
(80/20)
and
different electron acceptors.
Activities of methane
-
oxidizing consortia were tracked by
A
,
production of
13
C
-
dissolved inorganic carbon,
B
, concentration of sulfate, and
C
, concentration
of reduced AQDS (AQH
2
DS). One biological replicate was performed for each electron acceptor
condit
ion.
3
Supplementary Figure 2. Examples of paired phylogenetic identification and anabolic
activity of AOM consortia with different electron acceptors revealed by FISH
-
nanoSIMS.
A
,
D
,
G
,
J
, FISH images of AOM consortia with ANME in red and SRB in green. Sc
ale bar = 2
μm.
B
,
E
,
H
,
K
, Corresponding nanoSIMS ion images of
14
N
12
C
-
as a proxy for biomass, with bars
showing ion counts on the right.
C
,
F
,
I
,
L
, Single cell activity shown as
15
N atom percentage in
regions of interest (ROI). ANME ROIs are outlined in red and SRB ROIs are outlined in green.
Assimilation of
15
NH
4
+
into the biomass is used as a proxy for cellular anabolic activity, with
bars showing
15
N atom percentages on the right
.
4
Supplementary Figure
3
.
Correlation of ANME
-
2
anabolic activity and catabolic activity
after 9 days of incubation
.
The anabolic activity was measured by
15
N
-
ammonia incorporation
into
ANME
-
2a or ANME
-
2c consortia
(Supplementary Table 5). The catabol
ic activity was
measured by
13
C
-
dissolved inorganic carbon produced from
13
CH
4
oxidation
(Figure 1).
5
Supplementary Figure
4
. Average relative sulfur content of ANME
-
2a and ANME
-
2c
(
32
S/
12
C
14
N ANME) vs
their syntrophic
SRB partner (
32
S/
12
C
14
N
SRB).
ANME
-
SRB
consortia with different electron acceptors under CH
4
were evaluated after 9 days of incubation.
The sulfur content of
ANME or SRB
cells (
32
S/
12
C
14
N)
were
similar in our active incubations as
previously reported
[6]
.
T
he sulfur content of ANME showed no significant difference to that of
SRB
in the same consortium
in the 80 consortia analyzed (i.e. above or below the 1:1 line).
The
number of
consortia
analyzed in each electron acceptor condition can be found in Supplementary
Table
5
. Error bars represent standard deviation of the sulfur content of the cells in each
consortium.
Sulfate
AQDS
AQDS+Sulfate
None
Electron acceptors
1:1
6
Supplementary Figure
5
. The relationship between cell sulfur content and anabolism in
15
NH
4
+
incubations with different electron acceptors for ANME
-
2a and ANME
-
2c cells.
The
cellular
sulfur content
(
32
S/
12
C
14
N)
of
ANME
-
2a
cell ROIs
(n=2948) and ANME
-
2c
cell ROIs
(n=1225)
were
s
imilar in our active incubations as previously reported
[6]
.
However, in contrast
to the previous
report
, n
o significant correlation was found between the cell sulfur content and
anabolism
,
indicating that ANME is not
turning over sulfur proportional to their anabolic activity
(
15
N
-
ammoni
um
incorporation into the biomass). Cells are colored based on the
consortium
they
are from
. While
a few
consortia
showed
higher sulfur content than the rest
, the sulfur content of
the
consortia in the presence and in the absence of AQDS were not significantly different
(
AQDS+Sulfate
v.s. Sulfate: p=
0.19
for ANME
-
2a, p=
0.38
for ANME
-
2c; AQDS v.s. Sulfate:
p=0.39 for ANME
-
2a, p=0.28 for ANME
-
2c;
two
-
tailed t
-
test with equal variance
)
.
T
he number
of
cell
ROIs
analyzed in each electron acceptor condition can be found in Supplementary Table
4
.
7
Supplementary Figure
6
. Overview of metatranscriptome with different electron acceptors.
A
, Taxonomy assignment of mRNA reads based on NCBI Refseq database.
B
, Major metabolic
pathway assignment of mRNA reads based on KEGG database. Error bars represent standard
deviations of triplicate day 9 samples.
A)
B)
0
10
5
2
×
10
5
3
×
10
5
4
×
10
5
Methanosarcinales
Other Archaea
Deltaproteobacte
r
ia
Other Proteobacte
r
ia
Fi
r
micutes
Chlorofl
e
xi
Other Bacte
r
ia
Unassigned
T
r
ansc
r
ipts per million (TPM)
Condition
Sul
f
ate
A
QDS
A
QDS + Sul
f
ate
Methane Only
0
10
5
2
×
10
5
Methane metabolism
Sulfur metabolism
Carbon fixation
Cent
r
al carbo
h
yd
r
ate metabolism
L
ysine metabolism
Co
f
actor and vitamin biosynthesis
B
r
anched−chain amino acid metabolism
Pu
r
ine metabolism
Py
r
imidine metabolism
T
e
r
penoid backbone biosynthesis
Aromatic amino acid metabolism
T
r
ansc
r
ipts per million (TPM)
Condition
Sul
f
ate
A
QDS
A
QDS + Sul
f
ate
Methane Only
8
Supplementary Figure
7
. Propo
rtion of the non
-
rRNA transcripts accounted for by
metagenome
-
assembled genomes from the bulk metagenome.
9
Supplementary Figure
8
. Gene expression distributions of AOM consortia with different
electron acceptors after
3
days of incubation.
Normalized t
ranscript per million (TPM) was
used to compare RNA expression of
ANME
-
2 and
SEEP
-
SRB1 lineages
based on their
metagenome assembled g
enomes. Dashed lines indicate the quartiles of the distributions.
10
Supplementary Table 1. Summary of incubation sampling.
A total of 45
seep sediment
microcosms
with different headspace gas and electron acceptors
were setup on day 0.
A
,
S
upernatant from
the replicated
microcosms
were sampled for
geochemical
measurements
and
the number of replicates
used for each time point
in the analysis
is indicated in the table
.
B
, The
number of
microcosms
used for RNA
analysis.
A
day 0
day 3
day 6
day 9
CH
4
+sulfate
1
10
5
3
CH
4
+AQDS
1
10
6
3
CH
4
+sulfate+AQDS
1
10
6
3
CH
4
only
1
8
5
3
N
2
:C
O
2
+sulfate
1
N
2
:C
O
2
+AQDS
1
N
2
:C
O
2
+sulfate+AQDS
1
B
day 3
day 9
CH
4
+sulfate
1
3
CH
4
+AQDS
1
3
CH
4
+sulfate+AQDS
1
3
CH
4
only
1
3
Supplementary Table 2. Community composition analysis of sediment #7142 using
iTag
16S rRNA amplicon sequencing.
(separate table)
Supplementary Table 3. Statistics of the bulk metagenome and
metagenome
-
assembled
genomes.
Bulk
metagenome
ANME
-
2 cluster
archaeon S7142MS1
(ANME
-
2a)
ANME
-
2 cluster
archaeon S7142MS2
(ANME
-
2c)
Desulfobacterales
bacterium S7142MS3
(
SEEP
-
SRB1a)
Desulfobacterales
bacterium S7142MS4
(
SEEP
-
SRB1
g
)
Genome size (bp)
1813034
1647749
2766087
624626
Number of
scaffolds
247
387
820
239
GC content
45.97%
51.98%
41.19%
49.50%
Total gene count
2059
2022
2997
680
Genome
completeness
estimate
[7]
91.18%
90.38%
88.86%
36.28%
Genome
contamination
estimate
[7]
0.65%
8.20%
2.58%
0.16%
Strain
heterogeneity
[7]
0.00%
66.6
7%
25.00%
0.00%
Proportion of
sequence reads
4.3471%
1.2903%
4.0885%
0.1088%
GOLD Analysis
Project Id
Ga0401162
Ga0401164
Ga0401864
Ga0401165
Ga0401166
IMG Submission ID
221621
221617
222469
221619
221620
GenBank
accession number
PYCK00000000
PYCL00000000
JAABVG000000000
JAABVH000000000
11
Supplementary Table 4. FISH
-
nanoSIMS
analysis of ANME
-
SRB consortia at single
-
cell
level with different electron acceptors.
Electron acceptor
condition
Number of
cell
ROIs
a
nalyzed
ANME
-
2a
15
N
atom percent
ANME
-
2c
15
N
atom percent
SRB
15
N atom
percent
Sulfate
525
1.42 ± 0.39%
(n=219)
1.60 ± 0.46 (n=27)
1.97 ± 0.68
(n=279)
AQDS
2885
0.70 ± 0.43
(n=980)
0.80 ± 0.36
(n=516)
0.42 ± 0.12
(n=1389)
AQDS+Sulfate
3567
1.49 ± 0.45
(n=1204)
1.09 ± 0.33
(n=552)
0.52 ± 0.29
(n=1810)
None
1238
0.38 ± 0.02
(n=545)
0.38 ± 0.0002
(n=130)
0.37 ± 0.03
(n=563)
Supplementary Table 5. FISH
-
nanoSIMS analysis of ANME
-
SRB consortia at single
-
consortium level with different electron acceptors.
The mean of the
cell
ROIs in a consortium
was used for the
calculation.
Electron acceptor
condition
Number of
c
onsortia
a
nalyzed
ANME
-
2a
15
N
atom percent
ANME
-
2c
15
N
atom percent
SRB
15
N atom
percent
Sulfate
7
1.55 ± 0.41 (n=5)
1.61 ± 0.40 (n=2)
2.17 ± 0.87 (n=7)
AQDS
29
0.67 ± 0.36
(n=17)
0.88 ± 0.38 (n=12)
0.43 ± 0.09
(n=29)
AQDS+Sulfate
33
1.50 ± 0.32
(n=18)
1.15 ± 0.32 (n=15)
0.55 ± 0.18
(n=33)
None
11
0.38 ± 0.02 (n=6)
0.37 ± 0.02 (n=5)
0.38 ± 0.02
(n=11)
Supplementary Table 6. Key pathways in ANME
-
2a.
(separate
table)
Supplementary Table 7. Key pathways in ANME
-
2c.
(separate table)
Supplementary Table
8
. Differential gene expression
between AQDS
-
coupled and sulfate
-
coupled AOM
.
(separate table)
Supplementary Table
9
. Differential gene expression
during AQDS
-
coupled AOM
with or
without sulfate
amendment
.
(separate table)
12
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